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Description

TNF-alpha stimulated p110alpha or control siRNA transfected HUVECs previously labelled with celltracker orange dye were grown on collagen matrices in transwell chambers. Celltracker green-labelled THP-1 cells were added to HUVECs, and TEM allowed to proceed toward an MCP-1 gradient in the lower chamber, for either 10 or 60 minutes before fixation. Fixed samples were stained with AlexaFluor 633-conjugated phalloidin to visualize F-actin (grey) and z-stacks collected. Three dimensional reconstructions were produced using Volocity software. A small gamma correction was applied to the red channel in order to visualise weak cell tracker staining at the edges of cell, which are difficult to distinguish upon 3D rendering. Transwells were fixed by immersion in 3.7% (wt/vol) paraformaldehyde before being processed for immunofluorescence microscopy. Images were acquired at room temperature using a confocal microscope (LSM 510 META; Carl Zeiss, Inc.) with three single photomultiplier tube confocal detectors mounted on an inverted microscope (AxioObserver Z1; Carl Zeiss, Inc.) at a magnification of 40 with an oil immersion objective (EC Plan-Neofluar 40× NA 1.30 oil differential interference contrast M27). z sections were acquired at 200-nm intervals by confocal microscopy. Corresponds to Figure 5 from J Cell Biol. 2010 Mar 22;188(6):863-76.

Biological Sources

NCBI Organism Classification
Homo sapiens
(human)
Cell Type
blood vessel endothelial cell
monocytic leukemia cell
Cell Line
HUVEC
THP-1
Cellular Component
actin filament
cell

Biological Context

Biological Process
trans-endothelial migration

Attribution

Names
Robert J. Cain
Bart Vanhaesebroeck
Anne J. Ridley
Published
J Cell Biol. 2010 Mar 22;188(6):863-76.
Pubmed
20308428

Grouping

This image is part of a group.

Imaging

Image Type
recorded image
Imaging Mode
single-spot confocal microscopy
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
Visualization Methods
intracellular filling
phalloidin
Alexa Fluor 633
CellTracker Green
CellTracker Orange
Processing History
3D volume rendering
gamma correction

Sample Preparation

Methods
formaldehyde fixed tissue
Relation To Intact Cell
dispersed cells in vitro

Dimensions

Spatial Axis Image Size Pixel Size
X 512px 0.8789μm
Y 512px 0.8779μm
Z 79 2.5886μm
*CIL – Cell Image Library accession number. Please use this to reference an image.